Oral squamous cell carcinoma (OSCC) is the fifth most frequently occurring cancer worldwide. Common treatments still include radiation therapy and surgery leading to disfiguring impairments, which warrant less invasive treatments such as targeted therapies. Conditionally Replicative Adenoviruses (CRAds) are promising anti-cancer agents owing to their clinical safety and high infectivity. The anti-tumor effect of CRAds is based on specific killing of cancer cells by a natural lytic mechanism (oncolysis), which is distinct from that of antibody therapy and potentially allows overcoming antibody therapy resistance of recurrent and metastatic OSCC. The most recent genetic technology allows incorporation of a small affibody molecule with engineered targeting specificity into the Ad capsid protein fiber. We seek to pioneer implementation of this fiber modification technology in conjunction with a recently developed Epidermal Growth Factor Receptor (EGFR/ErbB1)-specific affibody for the development of a CRAd agent transductionally re-targeted to this cancer biomarker, overexpressed in most head and neck and oral cancers. Furthermore, the CRAd replication will be targeted to OSCC by CXCR4 Tumor Specific Promoter (TSP) with highly specific activation profile in oral cancers. On the other hand, a potentially limited intratumoral spread of the CRAd could be compensated by arming with a secretory protein MDA-7/IL24 gene, whose expression induces cell apoptosis in cancer-specific fashion via a bystander mechanism, fully compatible with oncolytic function of CRAd. In this application we also propose to perform an initial validation of the EGFR-targeted CXCR4-CRAd by testing its replication properties and oncolytic potential in the commonly used OSCC xenograft model in nude mice by a unique noninvasive multimodality imaging approach. Monitoring of tumor oncolysis via bioluminescence of OSCC tumor cells, stably expressing firefly luciferase and its correlation with intratumoral accumulation of red fluorescence, produced by the replicating CRAd with Infrared Fluorescent Protein 1.4 (IFP1.4)-labeled capsid, will provide an important tool for the initial assessment of the EGFR/OSCC dual targeting benefit. PUBLIC HEALTH RELEVANCE: The goal of the proposed research is to develop a new generation oncolytic adenovirus targeted to tumor marker EGFR/Erb1 for therapy and early detection of human head and neck and, particularly, oral cancers. The capsid of the new virus will be genetically tagged with a recently developed human EGFR-specific affibody for its transductional retargeting to EGFR. On the other hand, a new generation imaging reporter Infrared Fluorescent Protein 1.4 (IFP1.4) will be used to genetically label the viral particles to improve noninvasive real time imaging of viral replication and spread in tumors or biodistribution. We also propose to perform an initial targeting validation step of the new agent by imaging-based assessment of its replication in OSCC tumors and evaluation of its oncolytic efficacy in OSCC xenograft mouse model using a state-of-the-art multimodality imaging approach.